Nonalcoholic fatty liver disease (NAFLD) is a serious health problem worldwide. Since hepatic steatosis is closely associated with obesity and the metabolic syndrome, its frequency has greatly increased concomitantly with the worldwide pandemic of obesity. In obese individuals, chronically elevated serum free fatty acids (FFA) and high insulin levels lead to increased FFA uptake by the liver and increased synthesis of lipids resulting in hepatic triglyceride (TG) accumulation and insulin desensitization. The central hypothesis of this proposal is that obesity-associated NAFLD is primarily driven by the continuous protein- mediated uptake of fatty acids by the liver. If this assumption is correct, NAFLD in obese animals may be prevented, and even reversed by blocking the proteins thought to be responsible for hepatic insulin sensitivity and improve whole body glucose homeostasis. These assumptions can be tested by in vivo knockdown of liver FFA transporters (FATPs) and activators (ACSLs) using adeno-associated virus (AAV) mediate shRNA expression. Subsequently, further tests will indicate whether blocking FATPs and/or ACSLs can prevent and reverse diet-induced NAFLD. The specific aims of this project are: Aim 1: To establish in vivo knockdown of fatty acid transporters (FATPs) and acyl-CoA synthetases (ACSLs) using shRNA, which allow is less costly and less time intensive than using a knockout mouse model Aim 2: To characterize the contributions of FATPs and ACSLs to Fatty Liver Disease using non-invasive imaging technology such as micro-CT scanning and dual emission X-ray Aim 3: To investigate the impact of NAFLD reversal on while-body glucose homeostasis in obesity Taken together, these studies will provide new insights into the mechanisms of hepatic lipid homeostasis, the contributions of lipid-induced hepatic insulin desensitization to type-2 diabetes, and potential new targets for the treatment of NAFLD. Nonalcoholic fatty liver disease (NAFLD) is a serious health problem worldwide and is often associated with obesity and metabolic syndrome. This project suggests that hepatic steastosis and insulin desensitization in obese subjects is predominantly a result of protein-mediated uptake and activation of dietary and adipose tissue derived fatty acids by the liver, and that hepatic FATPs and ACSLs are required for this process and thus constitute targets for the treatment of obesity associated NAFLD. Ultimately, these experiments should clarify whether targeting hepatic FATPs and ACSLs, either via gene therapy or with small molecular inhibitors, is a viable approach to the treatment of NAFLD and insulin desensitization. [unreadable] [unreadable] [unreadable]